The present invention relates to a method and an apparatus for irradiating DNA marked with fluorescence or a fluorescent material with excitation light to inspect the DNA and a fluorescence detection method and more particularly to a method and an apparatus for detecting and inspecting DNA marked with a plurality of kinds of fluorescence samples or a plurality of kinds of fluorescent materials at high speed.
As a method for irradiating fluorescence-marked DNA with excitation light to inspect the DNA, there is a method for focusing laser light constituting excitation light on a sample as a single spot beam to detect fluorescence and collecting a fluorescence image by means of scanning of the excitation light and the sample. In detection of a plurality of fluorescent marks, a desired all area of a sample is first scanned with first excitation light to obtain a fluorescent image of a first fluorescent mark and the desired all area of the sample is then scanned with second excitation light again to collect the fluorescent image of a second fluorescent mark.
It is an object of the present invention to provide a high-speed detectable DNA inspection method and apparatus.
In the conventional method, a desired area is scanned with excitation light having different wavelength in accordance with a plurality of fluorescent marks and this scanning is repeated by the number of marks. In order to change a relative position of a single spot beam and a sample to inspect a desired area at high speed, it is necessary to move a stage on which the sample is placed at high speed. However, when the stage is moved at high speed, a considerable time is required for acceleration and deceleration for driving the stage since reciprocating motion is needed.
Further, when the scanning using the stage is repeatedly made for each wavelength of the excitation light, a positional shift or deviation occurs in a combined image of fluorescent images obtained by means of excitation lights when a driving accuracy of the stage and a reproduction accuracy are not sufficient. Consequently, high-speed and high-accuracy inspection of DNA required increasingly in future cannot be attained.
In order to solve the above problems, the present invention comprises measures described below.
A sample having a DNA piece added with a plurality of L kinds of fluorescence-marked materials combined to corresponding DNA is irradiated with a plurality of M kinds of minute spot excitation lights in accordance with the fluorescence-marked materials. Fluorescence intensities obtained in accordance with the fluorescence-marked materials are separately detected by means of the plurality of M kinds of minute spot excitation lights. The separately detecting operation of the plurality of fluorescent marks is made by changing a position on the sample irradiated with the spot excitation lights over a desired area by the number of times smaller than the number L of kinds of the fluorescence-marked materials, more preferably once, in order to detect the fluorescence intensities, to thereby inspect the DNA added with the plurality of kinds of fluorescence-marked materials.
Further, when the plurality of kinds of minute spot excitation lights are a plurality of minute multi-spot excitation lights, respectively, a plurality of points about the fluorescent marks can be detected at the same time to attain high-speed detection.
When different positions on the sample from one another are irradiated with the plurality of kinds of multi-spot excitation lights, fluorescence components from the fluorescent marks excited by the plurality of kinds of excitation lights can be separately detected with high accuracy without causing fluorescence components to impede one another as noise.
Irradiation with the plurality of kinds of multi-spot excitation lights is made at the same time and fluorescence obtained by the plurality of kinds of minute spot excitation lights in accordance with the fluorescence-marked materials is detected by a plurality of weak-light detection elements in accordance with respective excitation lights. The fluorescence intensities obtained in accordance with the fluorescence-marked materials are detected separately. Thus, in the photon-counting method used particularly when weak fluorescence is detected, the photon-counting can be made at the same time with respect to respective fluorescence components to make inspection at high speed in wide dynamic range.
Irradiation with the plurality of kinds of minute spot excitation lights is made in time series manner in accordance with wavelengths of the excitation lights and fluorescence obtained by the plurality of kinds of minute spot excitation lights in accordance with the fluorescence-marked materials is detected by a common weak-light detection element to respective excitation lights. The fluorescence intensities obtained in accordance with the fluorescence-marked materials are detected separately. Thus, in detection using the multi-spot, particularly, the weak-light detection element and its peripheral circuit become inexpensive.
Substantially the same position is irradiated with the plurality of kinds of minute spot excitation lights. Thus, detection by means of respective excitation lights can be made at the same place and detection with high accuracy can be made without any shift or deviation due to yawing and rolling of a stage in a combined image.
The plurality of kinds of minute spot excitation lights are turned on and off in different time zone within a time that a relative position of the spot excitation lights and the sample to be irradiated is changed by substantially one pixel. Thus, the respective fluorescent marks can be detected with high accuracy without mixing noise one another.
The plurality of kinds of minute spot excitation lights are changed stepwise at respective excitation light intensity levels within a time that a relative position of the spot excitation light and the sample to be irradiated is changed by substantially one pixel to detect the fluorescence intensity at each step, so that detection is made over a wide dynamic range at a high speed.
The DNA inspection apparatus according to the present invention is configured as follows. That is, the DNA inspection apparatus comprises one to a plurality of light sources for emitting lights having wavelengths different from one another, a plurality-of-wavelength excitation optical system for irradiating a DNA sample added with a plurality of fluorescence-marked materials with lights having the plurality of wavelengths from the light sources as minute spot excitation lights, a wavelength separation fluorescence detection system for separately detecting fluorescence intensities obtained by the respective excitation lights in accordance with the fluorescence-marked materials, a driving stage for changing a relative position of the minute spot excitation lights and the DNA sample over a desired area, and a processing unit for driving the driving stage so that the relative position of the minute spot excitation lights and the DNA sample is changed over the desired area by the number of times smaller than the number of kinds of the fluorescence-marked materials so as to construct image information of the plurality of fluorescence-marked DNAs on the sample from fluorescence detection information obtained by the detection system and the stage position information obtained by scanning by the number of times smaller than the number of kinds of the fluorescence-marked materials over the desired area.
Further, the present invention is applied to not only inspection of the fluorescence-marked DNA but also general fluorescent material emitting fluorescence peculiar to molecules such as protein.
According to the present invention, DNA to be inspected including the plurality of fluorescent marks or the samples containing the plurality of kinds of fluorescence materials cab be detected by scanning within a desired detection spread by the number of times smaller than the number of kinds of the samples containing the fluorescence materials or fluorescent marks, more preferably once, to thereby make it possible to attain detection extraordinarily fast as compared with the prior art. Consequently, a large number of objects to be inspected can be detected and inspected at high speed to attain largely temporal and economical effects.